The goal of this project is to investigate the function of Ku80 in tissue culture cells and in mice so that we may better understand those pathways that repair DNA double-strand breaks (DSBs) and monitor DNA damage. A Ku80 - Ku70 heterodimer forms the DNA binding component of DNA-dependent protein kinase (DNA-PK) and a third protein, DNA-PKcs, is the catalytic subunit. A deficiency in any one of thee proteins impairs the repair of DSBs that occur during V(D)J (variable [Diversity] Joining) recombination or after exposure to ionizing radiation. The scid (severe combined immune deficient) defect in mice was recently shown to be caused by a decrease in the level of DNA-PKcs. However, a null mutation has not been generated or observed for any of the genes that code for these proteins in mice, including scid. The gene that codes for Ku80, XRCC5, was mutated in mice by the embryonic stem cell/gene targeting technology. Mice are an ideal system to study DNA repair due to the remarkable similarity to humans and due to the sophisticated genetics. Preliminary results demonstrate the xrcc5 mutation is similar to the scid mutation; both result in immune deficient mice and g-radiation hypersensitive cell lines. However, unlike scid, the xrcc5 mutant mice are severely growth retarded and mutant cell lines proliferate slowly and age rapidly. This proposal will answer: What is the phenotype of xrcc5 mutant cell lines and mice? The specific aims are: 1) Analyze the phenotype of xrcc5 mutant mice. 2) Analyze cell proliferation, aging and radiation sensitivity in xrcc5 mutant cell lines. 3) Characterize the effects of an xrcc5 mutation on lymphocyte development and V(D)J recombination. 4) Analyze the repair of DSBs in mutant cell lines by end-to-end joining and nonhomologous and homologous recombination. Completion of these aims will significantly impact the way we think about the dynamic nature of DNA and may impact therapy for cancer and immune deficiency.